The mission of the Antibody Production and Characterization Core (APCC) is to produce and characterize antibodies for research use by members of the Breast Cancer Spore program. Core personnel are skilled in immunoassay development, hybridoma antibody and phage-display recombinant antibody technology, and methods for high-throughput antibody detection and characterization. The core uses traditional hybridoma technology to develop mouse and rat monoclonal antibodies and employs large rodent (about 2.9 x 10 to the 9th members) and human (about 5.9 x 10 to the 9th members) phage-displayed antibody libraries to obtain antigen-specific antibodies. The APCC has robotic systems (e.g. Genetix Q-Pix Colony-picker and Robbins HTS), 384- well visible and fluorescent plate readers, an imaging system (BioRad Fluors Max2), and a BIAcore 2000 for rapid antibody detection and characterization. The APCC has generated monoclonal and recombinant antibodies to more than seventy different antigens. The APCC has used phage-displayed recombinant antibody libraries to generate phospho-specific single chain fragment variable (ScFv) antibodies and will use this approach to address the specific aims of this grant. The Specific Aims of this proposal are initially to: 1) Utilize human or rodent phage antibody libraries to obtain recombinant antibodies specific for phospho-peptides representing domains on HER3 (erbB3) and p27 (project 1: Arteaga and Kelley), and Bcl-2, p21and cdc-25C (project 2: Pientenpol and Chak). 2) Develop immunological protocols (western blots, immunohistochemistry) for investigators to detect HER3, p21, p27, Bcl-2, and cdc-25C phosphorylated antigens in paraffin-embedded cells.